Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6489-6493, July 1995 Medical Sciences Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p2l (chromosomal loss/purine nucleoside phosphorylase/enzyme deficiency/CDKN2 gene) OLUFUNMILAYO I. OLOPADE*, HELEN M. POMYKALA, FITSUM HAGOS, LISE W. SVEEN, RAFAEL ESPINOSA III, MARTIN H. DREYLING, SUSAN GURSKY, WALTER M. STADLER, MICHELLE M. LE BEAU, AND STEFAN K. BOHLANDER Section of Hematology/Oncology, Department of Medicine and The University of Chicago Cancer Research Center, The University of Chicago Pritzker School of Medicine, Chicago, IL 60637 Communicated by Janet D. Rowley, The University of Chicago Pritzker School of Medicine, Chicago, IL, March 10, 1995 ABSTRACT Many human malignant cells lack methyl- similar protein, now called CDKN2B (p16INK4B), that shares thioadenosine phosphorylase (MTAP) enzyme activity. The 44% homology with CDKN2 in the first 50 amino acids and up gene (MTAP) encoding this enzyme was previously mapped to to 97% homology in the remainder of the protein (11). the short arm of chromosome 9, band p21-22, a region that is Whether CDKN2 is MLM remains unclear, because two recent frequently deleted in multiple tumor types. To clone candidate studies (12, 13) provide conflicting evidence. Hussussian et al. tumor suppressor genes from the deleted region on 9p2l-22, (12) described six disease-related germ-line mutations in we have constructed a long-range physical map of 2.8 mega- CDKN2 in 33 of 36 melanoma patients from nine families and bases for 9p2i by using overlapping yeast artificial chromo- suggested that CDKN2 likely is MLM. This is in contrast to 2 some and cosmid clones. This map includes the type IIFNgene of 13 mutations in 9p21-linked families and 0 of 38 familial cluster, the recently identified candidate tumor suppressor melanoma patients described by Kamb et al. (13). These genes CDKN2 (pl6INK4A) and CDKN2B (pl5INK4B), and several reports raise the possibility that CDKN2 may not be the only CpG islands. In addition, we have identified other transcrip- clinically relevant TSG on 9p and that loss of tumor suppres- tion units within the yeast artificial chromosome contig. sion may involve inactivation of other as yet unidentified genes Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG in in In island tbat maps between the IFN genes and CDKN2 reveals the region certain tumor types. support of this a predicted open reading frame of 283 amino acids followed by possibility Cheng et al. (14) published their findings of two 1302 nucleotides of 3' untranslated sequence. This gene is additional regions of nonoverlapping homozygous deletions on evolutionarily conserved and shows significant amino acid ho- 9p2l in malignant mesothelioma, one telomeric to CDKN2 and mologies to mouse and human purine nucleoside phosphorylases the other centromeric to it. and to a hypothetical 25.8-kDa protein in thepet gene (coding for The coincident loss of MTAP enzyme activity in many cytochrome bc, complex) region of Rhodospirilfum rubrum. The tumor cell lines with homozygous IFN gene deletions suggests location, expression pattern, and nucleotide sequence ofthis gene that MTAP is closely linked to the IFN gene cluster. We have suggest that it codes for the MTAP enzyme. suggested (15) that a 9p TSG should be localized between the IFN gene cluster and the MTAP locus based on IFN gene Unbalanced translocations or interstitial deletions of 9p are rearrangements seen in two cell lines and leukemia cells from recurring abnormalities in a variety of tumor types including one patient with deletions on 9p. In the reports published to acute lymphoblastic leukemia, glioma, melanoma, non-small date, it has been difficult to determine the exact position of the cell lung cancer, head and neck cancer, bladder cancer, and MTAP gene in relation to the homozygous deletions on 9p. mesothelioma (1). Homozygous deletion of DNA sequences In this manuscript, we describe the construction of a long- on 9p or loss of heterozygosity has now been described in range physical map around the IFN gene cluster that covers a multiple tumor types (2-7). A number of the cell lines and distance of 2.8 megabases (Mb), as determined by pulsed-field patient samples with 9p gene deletions also lack methylthio- gel electrophoresis (PFGE) and also the isolation of the MTAP adenosine phosphorylase (MTAP) enzyme activity. To our gene cDNA.t We have localized all of the known genes and knowledge, the gene encoding MTAP has not been cloned but several CpG islands on this map. Restriction sites and PFGE has been mapped to 9p22-9ql3 (8). In a few cases, the fragment sizes are clearly delineated on the resultant map, deletions that included both the IFN gene cluster and the which extends further proximally than the one presented by MTAP gene were interstitial and submicroscopic, suggesting Weaver-Feldhaus et al. (16). Also, several additional markers that these genes or a tumor suppressor gene (TSG) closely are described and localized on this map. The approximate linked to them was the target of the 9p deletions. This location of the shortest region of overlap of 9p deletions in hypothesis was further supported by the linkage of a gene that gliomas, melanomas, lung cancer, leukemia, mesothelioma, confers susceptibility to melanoma (MLM) to 9p2l in the head and neck cancer, and bladder cancers in relation to this region between D9S126 and the IFNA gene cluster (9). map is discussed. CDKN2 (p16INK4A) was recently proposed as a candidate TSG in this locus because the gene has been shown to be Abbreviations: YAC, yeast artificial chromosome; TSG, tumor sup- rearranged, deleted, or mutated in a majority of tumor cell pressor gene; Mb, megabase(s); STS, sequence tagged site; FISH, lines (6, 7). This gene codes for a 16-kDa protein (p16) that fluorescence in situ hybridization; SRO, shortest region of overlap; inhibits CDK4 and CDK6 by binding in competition with cyclin PNP, purine nucleoside phosphorylase; PFGE, pulsed-field gel elec- D (10). In humans, CDKN2 is adjacent to a gene encoding a trophoresis. *To whom reprint requests should be addressed at: The University of Chicago Medical Center, 5841 South Maryland Avenue, MC 2115, The publication costs of this article were defrayed in part by page charge Chicago, IL 60637-1470. payment. This article must therefore be hereby marked "advertisement" in tThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U22233). 6489 Downloaded by guest on September 26, 2021 6490 Medical Sciences: Olopade et al. Proc. Natl. Acad. Sci. USA 92 (1995) MATERIALS AND METHODS Screening for Expressed Sequences. The cosmids obtained by using probes from this ordered YAC were Cell Lines. The clinical and cytogenetic characterization of contig used for several different strategies to detect expressed sequences the tumor cell lines used in this study has been published (4, including direct screening of cDNA libraries, exon trapping, 5). Cell lines were used for the deletion mapping because they and a cDNA selection protocol based on the capture of provide an indefinite supply of DNA and the deletions present sequence independently amplified cosmid fragments by biotin- in cell lines were similar to the deletions observed in primary ylated cDNA (24, 25). Exon trapping was performed according leukemias and gliomas (2-4). to the manufacturer's protocol by using the Exon Trapping kit Analysis of Yeast Artificial Chromosome (YAC) Clones. (BRL/Life Technologies, Gaithersburg, MD). Each exon- to YAC clones corresponding IFNA1 (17, 18) and D9S966 (19) trapped product or cDNA-selected fragment from the cosmids sequence tagged sites (STSs) were isolated from the Centre was hybridized to a multiple-tissue Northern blot (Clontech), d'Etude Polymorphisme Humain (CEPH) MegaYAC library. a somatic cell hybrid panel (Oncor), "zoo blots" (Bios, New High molecular weight YAC DNA was isolated digested, Haven, CT), and Southern blots of tumor cell lines. Products electrophoresed, and blotted as described (17). To detect the that detected transcripts on Northern blot analysis were used left and right YAC vector arms, 346-bp HindIII-BamHI and to screen cDNA libraries from human adult brain, fetal brain, 276-bp BamHI-Sal I restriction fragments from the plasmid and fibroblast constructed in kgtlO or -11 (Clontech). In each pBR322 were used, respectively. Other probes used are as case, 7.5 x 105 plaques were screened. Positive clones were described in Fig. 1. The DNA probes were labeled with subcloned into the pBluescript vector (Stratagene) for se- [a-32P]dATP by using the random-primer labeling technique quencing. (20). PCR of the STSs was performed with 50 ng of YAC or Nucleotide Sequencing and Generation of STSs. Sequencing human genomic DNA as template. was performed on an Applied Biosystems model 373A DNA Fluorescence in Situ Hybridization (FISH) Analysis. YAC sequencing system with the Prism Ready Reaction Dye-Deoxy clones were purified by PFGE and the DNA was amplified by Terminator Cycle sequencing kit (Applied Biosystems). cDNA using a sequence-independent amplification technique (21). clones were sequenced entirely on both strands by using The amplification products were then labeled with biotin-11- double-stranded templates. The DNA sequence and the pre- dUTP or directly labeled nucleotides (Spectrum-Orange, Vy- dicted open reading frame were compared with GenBank data sis), and FISH was performed as described (22). bases (on September 13, 1994) by using the BLASTN and BLASTP Cloning YAC-End-Specific Clones. To obtain YAC-end- programs (26). specific probes, YAC-end rescue was performed as described by Hermanson et al. (23). Single-copy fragments from the YAC-end-rescued inserts were used as probes for Southern RESULTS blot hybridization to DNA of the different YACs and to a Construction of the Physical Map.